Biological substance quantitation method, pathological diagnosis support system and recording medium storing computer readable program

ABSTRACT

A biological substance quantitation method includes the following. A fluorescent image is input, which represents an expression of a specific biological substance in a sample stained with a fluorescent substance by a fluorescent bright spot. A quantitative evaluation value of the fluorescent bright spot is calculated. A standard fluorescent image of a standard sample stained under a same condition as the sample and representing an expression of the biological substance in a standard sample, is input under a same condition as the fluorescent image. A quantitative evaluation value in the standard fluorescent image is calculated under a same condition as the fluorescent image. Based on a correlation between an expression amount of the biological substance in a standard sample measured in advance and the evaluation value in the standard fluorescent image, the evaluation value in the fluorescent image is converted to an expression amount of the biological substance in the sample.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a 371 of PCT/JP2015/053679 filed on Feb. 10, 2015,which is incorporated herein by reference.

TECHNOLOGICAL FIELD

The present invention relates to a method for quantitating a biologicalsubstance, a pathological diagnosis support system, and a program usingluminance information of a fluorescent substance.

BACKGROUND ART

In recent years, with the spread of therapy using molecular target drugsbased mainly on antibody drugs, quantitating a biological substance incells of the observation target has been desired for more efficientdesign of the molecular target drugs. For confirming the presence of aspecific biological substance, a method of tissue analysis is known onthe basis of staining of cells using a fluorescent substancespecifically bindable to the biological substance.

Patent Document 1 proposes a method of improving accuracy inquantitating the expression amount of biological substance by measuringthe number of fluorescent bright spot in the tissue sample as follows:staining the tissue sample with a phosphor bindable to the biologicalsubstance, analyzing peaks in the luminance distribution of thefluorescent bright spot in the tissue sample, and calculating averageluminance of one particle of the phosphor.

PRIOR ART DOCUMENT Patent Document

Patent Document 1: Japanese Patent Application Laid-Open Publication No.2013-57631

SUMMARY Problems to be Solved by the Invention

However, it is generally known that errors often occur in quantitationresult of a biological substance from a stained tissue sample due to,for example, variations in staining conditions (for example, reactiontime, concentration of solvent, and temperature), different operator, ordifferent measurement system (for example, measuring instrument such asmicroscope and camera).

According to the conventional technique described in patent document 1,there is a problem that the analysis result often varies due todifferent operator or different measurement system in performingcomparison analysis of quantitation results of a biological substanceobtained from a plurality of samples.

In a pathological diagnosis, it is desired that evaluation by comparingquantitation results is possible not only between samples from whichbiological substance quantitation is performed at the same time but alsobetween samples obtained by different operator, different measurementinstrument, or different staining conditions, i.e., the diagnosis resultof one patient is desired to be compared before after conductingtreatment.

A main object of the present invention is to provide a biologicalsubstance quantitation method and a program for accurate quantitation ofa specific biological substance in a sample by correcting errors causedby the difference in operator and measurement system.

Means for Solving the Problem

To achieve at least one of the abovementioned objects, according to anaspect of the present invention, there is provided a biologicalsubstance quantitation method which quantitates an expression amount ofa specific biological substance in a sample stained with a stainingreagent which stains the biological substance with a fluorescentsubstance, the method including:

-   -   inputting a fluorescent image which represents an expression of        the biological substance in the sample by a fluorescent bright        spot;    -   performing fluorescence quantitation which includes calculation        of an evaluation value by quantitative evaluation of the        fluorescent bright spot in the fluorescent image;    -   inputting a standard fluorescent image under a same condition as        the inputting of the fluorescent image, wherein        -   the standard fluorescent image represents an expression of            the biological substance in a standard sample by a            fluorescent bright spot based on staining under a same            condition as the sample, and        -   an expression amount of the biological substance in the            standard sample is measured in advance;    -   performing standard fluorescence quantitation which includes        calculation of an evaluation value by quantitative evaluation of        the fluorescent bright spot in the standard fluorescent image        under a same condition as the quantitation of fluorescence;    -   calculating a correlation between the expression amount of the        biological substance in the standard sample and the evaluation        value of the fluorescent bright spot in the standard fluorescent        image; and    -   converting the evaluation value of the fluorescent bright spot        in the fluorescent image to an expression amount of the        biological substance in the sample based on the calculating of        the correlation.

The invention of claim 2 provides the biological substance quantitationmethod according to claim 1, wherein

-   -   a calibration curve is prepared in calculating the correlation,        wherein the calibration curve represents the evaluation value of        the fluorescent bright spot of the standard fluorescence image        corresponding to the expression amount of the biological        substance in the standard sample, and    -   in the converting, the evaluation value of the fluorescent        bright spot of the fluorescent image is converted to the        expression amount of the biological substance in the sample        based on the calibration curve.

The invention of claim 3 provides the biological substance quantitationmethod according to claim 1 or 2, wherein the standard sample is a cellcultured on a substrate.

The invention of claim 4 provides the biological substance quantitationmethod according to any one of claims 1 to 3, wherein the biologicalsubstance is a protein.

The invention of claim 5 provides the biological substance quantitationmethod according to any one of claims 1 to 4, wherein the stainingreagent includes a fluorescent particle in which a plurality ofmolecules of the fluorescent substance are accumulated.

To achieve at least one of the abovementioned objects, according toanother aspect of the present invention, there is provided apathological diagnosis support system which quantitates an expressionamount of a specific biological substance in a sample stained with astaining reagent which stains the biological substance with afluorescent substance, the system including:

-   -   a fluorescent image inputting unit which inputs a fluorescent        image which represents an expression of the biological substance        in the sample by a fluorescent bright spot;    -   a fluorescence quantitation unit which calculates an evaluation        value by quantitative evaluation of the fluorescent bright spot        in the fluorescent image;    -   a standard fluorescent image inputting unit which inputs a        standard fluorescent image under a same condition as the        fluorescent image inputting unit, wherein        -   the standard fluorescent image represents an expression of            the biological substance in a standard sample by a            fluorescent bright spot based on staining under a same            condition as the sample, and        -   an expression amount of the biological substance in the            standard sample is measured in advance;    -   a standard fluorescent quantitation unit which calculates an        evaluation value by quantitative evaluation of the fluorescent        bright spot in the standard fluorescent image under a same        condition as the fluorescence quantitation unit;    -   a correlation calculator which calculates a correlation between        the expression amount of the biological substance in the        standard sample and the evaluation value of the fluorescent        bright spot in the standard fluorescent image; and    -   a converter which converts the evaluation value of the        fluorescent bright spot in the fluorescent image to an        expression amount of the biological substance in the sample        based on the correlation.

To achieve at least one of the abovementioned objects, according toanother aspect of the present invention, there is provided a programcausing a computer which quantitates an expression amount of a specificbiological substance in a sample stained with a staining reagent whichstains the biological substance with fluorescent substances, to functionas:

-   -   a fluorescent image inputting unit which inputs a fluorescent        image which represents an expression of the biological substance        in the sample by a fluorescent bright spot;    -   a fluorescence quantitation unit which calculates an evaluation        value by quantitative evaluation of the fluorescent bright spot        in the fluorescent image;    -   a standard fluorescent image inputting unit which inputs a        standard fluorescent image under a same condition as in the        fluorescent image inputting unit, wherein        -   the standard fluorescent image represents an expression of            the biological substance in a standard sample by a            fluorescent bright spot based on staining under a same            condition as the sample, and        -   an expression amount of the biological substance in the            standard sample is measured in advance;    -   a standard fluorescent quantitation unit which calculates an        evaluation value by quantitative evaluation of the fluorescent        bright spot in the standard fluorescent image under a same        condition as the fluorescence quantitation unit;    -   a correlation calculator which calculates a correlation between        the expression amount of the biological substance in the        standard sample and the evaluation value of the fluorescent        bright spot in the standard fluorescent image; and    -   a converter which converts the evaluation value of the        fluorescent bright spot in the fluorescent image to an        expression amount of the biological substance in the sample        based on the correlation.

Advantageous Effects of Invention

According to the present invention, the amount of a specific biologicalsubstance in a sample can be accurately quantitated.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features provided by one or more embodiments of theinvention will become more fully understood from the detaileddescription given hereinbelow and the appended drawings which are givenby way of illustration only, and thus are not intended as a definitionof the limits of the present invention.

FIG. 1 is a diagram showing a configuration of a pathological diagnosisassistance system using the biological substance quantitation methodaccording to the present invention;

FIG. 2 is a block diagram showing a functional configuration of an imageprocessing device in FIG. 1;

FIG. 3 is a diagram illustrating an exemplary bright field image;

FIG. 4 is a diagram illustrating an exemplary fluorescent image;

FIG. 5 is a flowchart illustrating steps for biological substancequantitation executed in the pathological diagnosis assistance systemaccording to the present invention;

FIG. 6 is a flowchart illustrating detailed process in Step S4 of FIG.5;

FIG. 7A is a diagram illustrating an exemplary bright field image;

FIG. 7B is a diagram illustrating an image (cell image) of cell regionsextracted from the bright field image in FIG. 7A.

FIG. 8A is a diagram illustrating an exemplary fluorescent image;

FIG. 8B is a diagram illustrating an image (bright point image) ofbright spot regions extracted from the fluorescent image in FIG. 8A.

FIG. 9 is a diagram illustrating exemplary calibration curves usingdifferent concentrations of staining reagent.

EMBODIMENT FOR CARRYING OUT THE INVENTION

Embodiments for carrying out the present invention will now be describedwith reference to the attached drawings, which should not be construedto limit the present invention.

<Configuration of Pathological Diagnosis Support System 100>

FIG. 1 illustrates an exemplary overall configuration of a pathologicaldiagnosis support system 100 that employs the quantitative determinationmethod of a biological substance according to the present invention. Thepathological diagnosis support system 100 acquires microscopic images ofa sample of observation target (hereinafter referred to as a “targetsample”) and a sample in which concentration of a specific biologicalsubstance is quantitated in advance (hereinafter referred to as a“standard sample”), analyzes the acquired microscopic images, andoutputs a feature quantity which quantitatively represents expression ofthe specific biological substance in the target sample of observationtarget.

As illustrated in FIG. 1, the pathological diagnosis support system 100includes a microscopic image acquiring device 1A, an image processingdevice 2A, and an interface, such as a cable 3A, connecting themicroscopic image acquiring device 1A and the image processing device 2Afor transmission and reception of data. The microscopic image acquiringdevice 1A may be connected to the image processing device 2A in anymanner. For example, the microscopic image acquiring device 1A and theimage processing device 2A may be connected through a local area network(LAN) or wireless communication.

The microscopic image acquiring device 1A is a known optical microscopeprovided with a camera, which acquires a microscopic image of a tissuesample on a microscopic slide placed on a slide fixation stage, andtransmits the microscopic image to the image processing device 2A.

The microscopic image acquiring device 1A includes an irradiator, afocusing unit, a photographing unit, and a communication interface(I/F). The irradiator includes a light source and a filter, and emitslight toward the tissue sample on the microscopic slide placed on theslide fixation stage. The focusing unit includes an eyepiece lens and anobject lens. The focusing unit focuses transmitted light, reflectedlight, or fluorescent light, which is emitted from the tissue sample onthe microscopic slide in response to the irradiated light, into animage. The photographing unit includes a charge coupled device (CCD)sensor. The photographing unit is specifically a camera disposed in amicroscope to photograph an image formed by the focusing unit, andproduce the digital image data of the microscopic image. Thecommunication interface transmits the image data of the microscopicimage to the image processing device 2A. The microscopic image acquiringdevice 1A in the present embodiment includes a bright field unitsuitable for bright field microscopy composed of a combination of anirradiating subunit and a focusing subunit, and a fluorescence unitsuitable for fluorescent microscopy composed of a combination of anirradiating subunit and a focusing subunit, and can switch between theseunits, i.e., between bright field observation and fluorescenceobservation.

Besides the microscope provided with a camera, the microscopic imageacquiring device 1A may be any device, for example, a virtualmicroscopic slide preparing device that scans a microscopic slide placedon a slide fixation stage of a microscope to acquire a microscopic imageof an overall tissue sample (see Japanese Publication of InternationalPatent Application No. 2002-514319, for example). The virtualmicroscopic slide preparing device can acquire image data of the overalltissue sample that can be displayed on a display at once.

The image processing device 2A analyzes the microscopic imagetransmitted from the microscopic image acquiring device 1A to calculatethe distribution of the expression of the specific biological substancein the target tissue sample.

FIG. 2 illustrates an exemplary functional configuration of the imageprocessing device 2A. As illustrated in FIG. 2, the image processingdevice 2A includes a controller 21, an operating unit 22, a display 23,a communication interface 24, and a storage 25, which are connected toeach other through a bus 26.

The controller 21 includes a central processing unit (CPU), a randomaccess memory (RAM), and the like. The controller 21 executes multipleprocesses in cooperation with a variety of programs stored in thestorage 25 to control the overall operation of the image processingdevice 2A. For example, the controller 21 executes image analysis incooperation with a program stored in the storage 25 (see steps S4 to S6in FIG. 5) to and functions as a unit executing a fluorescentquantitation step, standard fluorescence quantitation step, correlationcalculation step, and conversion step.

The operating unit 22 includes a keyboard including keys for inputtingcharacters and numbers and several functional keys, and a pointingdevice, such as a mouse. The operating unit 22 outputs input signals tothe controller 21, i.e., signals generated by press of keys on thekeyboard and by operation of the mouse.

The display 23 includes a monitor, such as a cathode ray tube (CRT)display or a liquid crystal display (LCD). The display 23 displays avariety of windows in response to display signals input from thecontroller 21.

The communication interface 24 allows data transmission and receptionbetween the microscopic image acquiring device 1A and external devices,such as the microscopic image acquiring device 1A. The communicationinterface 24 functions as an input unit of inputting a standardfluorescent image and a fluorescent image.

The storage 25 includes a hard disk drive (HDD) or a nonvolatile memorycomposed of a semiconductor, for example. The storage 25 stores avariety of programs and data as described above.

Besides, the image processing device 2A may include a LAN adaptor and arouter to be connected to external devices through a communicationnetwork, such as a LAN.

The image processing device 2A in the present embodiment preferablyanalyzes the sample using the bright field image and fluorescent imagetransmitted from the microscopic image acquiring device 1A.

The bright field image is a microscopic image of a sample stained with ahematoxylin (H) staining reagent or a hematoxylin-eosin (HE) stainingreagent focused and photographed in the bright field with themicroscopic image acquiring device 1A. The bright field image representsmorphology of cells in the sample. The hematoxylin is a blue violet dyefor staining basophilic tissues, such as cell nuclei, bone tissues, partof cartilaginous tissues, and serum components. The eosin is a red topink color dye for staining acidophilic tissues, such as cytoplasms,connective tissues of soft tissues, erythrocytes, fibrin, and endocrinegranules. FIG. 3 illustrates an exemplary bright field image of anHE-stained tissue sample.

The fluorescent image is a microscopic image obtained as follows: Asample is stained with a staining reagent containing a fluorescentsubstance bonded to a biological substance recognition site whichspecifically bonds or reacts with the specific biological substance. Thesample is irradiated with an excitation light having a predeterminedwavelength in the microscopic image acquiring device 1A so that thefluorescent substance emits light (fluorescent light). The fluorescentlight is enlarged, focused, and photographed. In other words, thefluorescent light in the fluorescent image represents the expression ofthe specific biological substance corresponding to the biologicalsubstance recognition site in the sample. FIG. 4 illustrates anexemplary fluorescent image.

<Acquisition of Fluorescent Image>

A method of acquiring the fluorescent image will now be described indetail, including the staining reagent used in acquisition of afluorescent image and the method of staining a sample with the stainingreagent.

[Fluorescent Substance]

Examples of the fluorescent substance used as the staining reagent foracquiring a fluorescent image include an organic fluorescent dye and aquantum dot (a semiconductor particle). The fluorescent substancespreferably emit a visible light to a near-infrared light having awavelength in the range of 400 to 1100 nm when excited by an ultravioletlight to a near-infrared light having a wavelength in the range of 200to 700 nm.

Examples of the organic fluorescent dyes include fluorescein dyemolecules, rhodamine dye molecules, Alexa Fluor (registered trademark,made by Invitrogen Corporation) dye molecules, BODIPY (registeredtrademark, made by Invitrogen Corporation) dye molecules, cascading dyemolecules, coumarin dye molecules, eosin dye molecules, NBD dyemolecules, pyrene dye molecules, Texas Red dye molecules, and cyaninedye molecules.

Specific examples thereof include 5-carboxy-fluorescein,6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein,6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein,6-carboxy-2′,4,7,7′-tetrachlorofluorescein,6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, naphthofluorescein,5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine,rhodamine 6G, tetramethylrhodamine, X-rhodamine, Alexa Fluor 350, AlexaFluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, AlexaFluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, AlexaFluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, AlexaFluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665 (made by InvitrogenCorporation), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, and Cy7.These organic fluorescent dyes may be used alone or in combination.

The quantum dot may contain Group II-VI compounds, Group III-Vcompounds, or Group IV elements as a component (also referred to as a“Group II-VI quantum dot”, “Group III-V quantum dot”, or “Group IVquantum dot”, respectively) can be used. These quantum dots may be usedalone or in combination.

Specific examples thereof include, but should not be limited to, CdSe,CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, andGe.

[Fluorescent Substance-Encapsulating Nanoparticle]

The fluorescent substance used as a staining reagent for obtaining afluorescent image in the present embodiment may be a fluorescentsubstance-encapsulating nanoparticle (hereinafter, referred to as afluorescent particle) in which a plurality of molecules of thefluorescent substance are accumulated. The fluorescent particle refersto a nanoparticle in which the fluorescent substance is dispersed. Thefluorescent substance and the nanoparticle may or may not be chemicallybonded with each other. The material composing the nanoparticle is notparticularly limited, and examples thereof include silica, polystyrene,polyactate acid, melamine, and the like.

A quantum dot having a core of a quantum dot and an outer shell may beused as a fluorescent particle. Throughout the specification, thequantum dot having a shell is represented, for example, as CdSe/ZnSwhere the core is CdSe and the shell is ZnS. Examples of usable quantumdots having a core of a quantum dot and a shell include, but should notbe limited to, CdSe/ZnS, CdS/ZnS, InP/ZnS, InGaP/ZnS, Si/SiO₂, Si/ZnS,Ge/GeO₂, Ge/ZnS.

A quantum dot having a surface treated with an organic polymer may alsobe used when necessary. Examples of such quantum dots include CdSe/ZnShaving surface carboxy groups (made by Invitrogen Corporation), andCdSe/ZnS having surface amino groups (made by Invitrogen Corporation).

The fluorescent particle used in the present embodiment can be preparedby any known method. Encapsulation of a fluorescent dye in thenanoparticle may be performed by any method, for example, particlesynthesis by bonding a fluorescent dye molecule to a raw-materialmonomer, or introduction of a fluorescent dye by adsorbing into theresin.

For example, a polystyrene nanoparticle encapsulating an organicfluorescent dye can be prepared by a copolymerization process using anorganic dye having a polymerizable functional group as described in U.S.Pat. No. 4,326,008 (1982), or by impregnation of a polystyrenenanoparticle with an organic fluorescent dye as described in U.S. Pat.No. 5,326,692 (1992).

A polymer nanoparticle encapsulating quantum dots can be prepared byimpregnation of a polystyrene nanoparticle with quantum dot, which isdisclosed in Nature Biotechnology vol. 19, p. 631 (2001).

The fluorescent particle used in the present embodiment may have anyaverage particle size, but is preferably 40 to 280 nm. Too large averageparticle size readily results in saturation of luminance and inaccuratemeasurement when fluorescent particles are close to each other. When theaverage particle size is small, integrated value of luminance of onefluorescent particle is small and the fluorescent signal is easilyburied in background noises (noises of camera or autofluorescence ofcells).

The average particle size is determined as follows: Cross-sectional areaof each particle is measured in an electron microscopic photograph takenwith a scanning electron microscope (SEM). The observed area of eachparticle is regarded as the area of a circle, and the diameter of thecircle is determined as the particle size. In the present application,the sizes of 1000 particles are measured, and the arithmetic average ofthem is determined as the average particle size.

[Binding of Biological Substance Recognition Site to FluorescentParticle]

The biological substance recognition site according to the presentembodiment is a site specifically bindable and/or reactive to a targetbiological substance. The target biological substance may be anybiological substance specifically bindable to the site. Typical examplesof the target biological substance include proteins (peptides), nucleicacids (oligonucleotides, polynucleotides), and antibodies. Accordingly,examples of a substance specifically bindable to the target biologicalsubstance include antibodies that can recognize the proteins asantigens, other proteins specifically bindable to the proteins, andnucleus acids having base sequences allowing hybridization to thenucleus acids. Specific examples thereof include an anti-HER2 antibodyspecifically bindable to HER2 or a protein to be expressed on surfacesof cells; a Ki67 antibody specifically bindable to Ki67 protein as acell proliferation marker to be expressed in cell nuclei; an anti-ERantibody specifically bindable to an estrogen receptor (ER) to beexpressed in cell nuclei; and an anti-actin antibody specificallybindable to actin that forms a cell skeleton. Among these antibodies,the anti-HER2 antibody, the anti-ER antibody, or the anti-Ki67 antibodyis preferred because a fluorescent particle bonded to them can be usedin selection of drugs for breast cancer.

Examples of the specific antigens include the followings. The antibodiesfor recognizing these antigens are commercially available from a varietyof antibody manufacturers, and can also be produced based on knowledgegenerally shared. Examples of the specific antigens include M. actin,M.S. actin, S.M. actin, ACTH, Alk-1, α1-antichymotrypsin,α1-antitrypsin, AFP, bcl-2, bcl-6, β-catenin, BCA 225, CA19-9, CA125,calcitonin, calretinin, CD1a, CD3, CD4, CD5, CD8, CD10, CD15, CD20,CD21, CD23, CD30, CD31, CD34, CD43, CD45, CD45R, CD56, CD57, CD61, CD68,CD79a, “CD99, MIC2”, CD138, chromogranin, c-KIT, c-MET, collagen typeIV, Cox-2, cyclin D1, keratin, cytokeratin (high molecular weight),pan-keratin, pan-keratin, cytokeratin 5/6, cytokeratin 7, cytokeratin 8,cytokeratin 8/18, cytokeratin 14, cytokeratin 19, cytokeratin 20, CMV,E-cadherin, EGFR, ER, EMA, EBV, factor VIII-related antigen, fascin,FSH, galectin-3, gastrin, GFAP, glucagon, glycophorin A, granzyme B,hCG, hGH, Helicobacter pylori, HBc antigen, HBs antigen, hepatocytespecific antigen, HER2, HSV-I, HSV-II, HHV-8, IgA, IgG, IgM, IGF-1R,inhibin, insulin, kappa L chain, Ki67, lambda L chain, LH, lysozyme,macrophage, melan A, MLH-1, MSH-2, myeloperoxidase, myogenin, myoglobin,myosin, neurofilament, NSE, p27 (Kip1), p53, p53, P63, PAX 5, PLAP,Pneumocystis carinii, Podoplanin (D2-40), PGR, prolactin, PSA, prostaticacid phosphatase, renal cell carcinoma, S100, somatostatin, spectrin,synaptophysin, TAG-72, TdT, thyroglobulin, TSH, TTF-1, TRAcP, tryptase,bilin, vimentin, WT1, and Zap-70.

In the case where the target biological substance is a nucleus acid, thefollowing specific nucleus acid genes whose relations with diseases arepointed out can be exemplified. Probes recognizing these specificnucleus acid genes are commercially available as BAC probes, and canalso be produced based on knowledge generally shared. Specific examplesof the specific nucleus acid genes are listed below. Examples of genesrelated to proliferation of cancer or response rates of molecular targetdrugs include HER2, TOP2A, HER3, EGFR, P53, and MET. Known examples ofcancer related genes are as follows. Examples of tyrosine kinase relatedgenes include ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS(CSF1R), DDR2,EGFR(ERBB1), HER4(ERBB4), EML4-ALK, IGF1 R, EPHA1, INSR, EPHA2,IRR(INSRR), EPHA3, KIT, EPHA4, LTK, EPHA5, MER(MERTK), EPHA6, MET,EPHA7, MUSK, EPHA8, NPM1-ALK, EPHB1, PDGFRα(PDGFRA), EPHB2,PDGFRβ(PDGFRB)EPHB3, RET, EPHB4, RON(MST1R), FGFR1, ROS(ROS1), FGFR2,TIE2(TEK), FGFR3, TRKA(NTRK1), FGFR4, TRKB(NTRK2), FLT1(VEGFR1), andTRKC(NTRK3). Examples of breast cancer related genes include ATM, BRCA1,BRCA2, BRCA3, CCND1, E-Cadherin, ERBB2, ETV6, FGFR1, HRAS, KRAS, NRAS,NTRK3, p53, and PTEN. Examples of genes related to carcinoid tumorsinclude BCL2, BRD4, CCND1, CDKN1A, CDKN2A, CTNNB1, HES1, MAP2, MEN1,NF1, NOTCH1, NUT, RAF, SDHD, and VEGFA. Examples of colorectal cancerrelated genes include APC, MSH6, AXIN2, MYH, BMPR1A, p53, DCC, PMS2,KRAS2 (or Ki-ras), PTEN, MLH1, SMAD4, MSH2, STK11, and MSH6. Examples oflung cancer related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A,RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, and MYC. Examples of livercancer related genes include Axin1, MALAT1, b-catenin, p16 INK4A,c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1,and KRAS. Examples of kidney cancer related genes include Alpha, PRCC,ASPSCR1, PSF, CLTC, TFE3, p54nrb/NONO, and TFEB. Examples of thyroidcancer related genes include AKAP10, NTRK1, AKAP9, RET, BRAF, TFG, ELE1,TPM3, H4/D10S170, and TPR. Examples of ovarian cancer related genesinclude AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA,GATA4, RB, HRAS, RET, KRAS, and RNASET2. Examples of prostate cancerrelated genes include AR, KLK3, BRCA2, MYC, CDKN1B, NKX3.1, EZH2, p53,GSTP1, and PTEN. Examples of bone tumor related genes include CDH11,COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.

The biological substance recognition site may be bonded to a fluorescentparticle with any bond. Examples of the bonding form include covalentbond, ionic bond, hydrogen bond, coordination bond, physical adsorption,and chemical adsorption. Bonds having strong forces, such as covalentbond, are preferred in view of stability of the bond.

An organic molecule may link between the biological substancerecognition site and the fluorescent particle. For example, apoly(ethylene glycol) chain, such as SM(PEG)12 made by Thermo ScientificInc., may be used to inhibit non-specific adsorption of a biologicalsubstance.

For example, the biological substance recognition site is bonded tofluorescent substance-encapsulating a silica nanoparticle according tothe same procedure in both the fluorescent substance composed of anorganic fluorescent dye and that composed of a quantum dot. For example,the biological substance recognition site may be bonded to a fluorescentsubstance-encapsulating silica nanoparticle with a silane couplingagent, which is widely used in bonding an inorganic substance to anorganic substance. The silane coupling agent has an alkoxysilyl group atone terminal of the molecule to yield a silanol group throughhydrolysis, and has a functional group, such as a carboxyl, amino,epoxy, or aldehyde group, at the other terminal. The silane couplingagent binds to an inorganic substance through an oxygen atom of thesilanol group. Specific examples thereof includemercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane,aminopropyltriethoxysilane, and silane coupling agents having apoly(ethylene glycol) chain (such as PEG-silane no. SIM6492.7 made byGelest, Inc.). Two or more silane coupling agents may be used incombination.

The organic fluorescent dye-encapsulating nanoparticle may be reactedwith a silane coupling agent according to a known procedure. Forexample, the organic fluorescent dye-encapsulating nanoparticle isdispersed in pure water, and aminopropyltriethoxysilane is added to bereacted with the particle at room temperature for 12 hours. After thereaction is completed, the product is centrifuged or filtered to yieldorganic fluorescent dye-encapsulating nanoparticle having a surfacemodified with an aminopropyl group. The amino group can be reacted witha carboxyl group in an antibody, so that the antibody is bonded to theorganic fluorescent dye-encapsulating nanoparticle through an amidobond. A condensing agent, such as EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride: availablefrom Pierce (registered trademark)), may also be used when necessary.

A linker compound having a site that can be directly bonded to anorganic fluorescent dye-encapsulating nanoparticle modified with anorganic molecule and a site that can be bonded to a molecular targetsubstance may be used when necessary. Specifically, in use of sulfo-SMCC(sulfosuccinimidyl 4[N-maleimidomethyl]-cyclohexane-1-carboxylate:available from Pierce) having both a site selectively reactive with anamino group and a site selectively reactive with a mercapto group, theamino group of the organic fluorescent dye-encapsulating nanoparticlemodified with aminopropyltriethoxysilane can be bonded to a mercaptogroup in the antibody, so that an organic fluorescent dye-encapsulatingnanoparticle bonded with an antibody is formed.

When the biological substance recognition site is bonded to eachfluorescent substance-encapsulating polystyrene nanoparticle, the sameprocedure can be used both in the case where the fluorescent substanceis an organic fluorescent dye and in the case where the fluorescentsubstance is a quantum dot. In other words, impregnation of apolystyrene nanoparticle having a functional group, such as an aminogroup, with an organic fluorescent dye or a quantum dot can yield afluorescent substance-encapsulating polystyrene nanoparticle having thefunctional group. Use of EDC or sulfo-SMCC in the subsequent step canyield a fluorescent substance-encapsulating polystyrene nanoparticlehaving an antibody.

[Staining Process]

The method of staining a tissue sample will now be described. Samplesprepared by any known method can be applied to the staining processdescribed below.

The quantitation method according to the present invention can beapplied not only to a paraffin-embedded tissue sample but also to a cellsample fixed onto a substrate and the like.

1) Deparaffinizing Step

A tissue sample is immersed in xylene in a vessel to remove paraffin atany temperature, for example, at room temperature. A preferred immersiontime is 3 minutes or more and 30 minutes or less. Xylene may be replacedwith fresh one during the immersion as needed.

The tissue sample is then immersed in ethanol in a vessel to removexylene. The immersion may be performed at any temperature, for example,at room temperature. A preferred immersion time is 3 minutes or more and30 minutes or less. Ethanol may be replaced with fresh one during theimmersion as needed.

The tissue sample is then immersed in water in a vessel to removeethanol at any temperature, for example, at room temperature. Apreferred immersion time is 3 minutes or more and 30 minutes or less.Water may be replaced with fresh one during the immersion as needed.

2) Retrieval Process

A target biological substance is retrieved by a known process. Theretrieval may be performed under any condition, and a solution forretrieval may be, for example, a 0.01M citric acid buffer solution (pH:6.0), a 1 mM EDTA solution (pH: 8.0), 5% urea, or a 0.1Mtrishydrochloric acid buffer solution. An autoclave, a microwave, apressure pan, or a water bath may be used as a heater. The retrieval maybe performed at any temperature, for example, at room temperature. Thesample may be retrieved at a temperature of 50 to 130° C. for 5 to 30minutes.

The activated sample is then immersed in phosphate buffered saline (PBS)in a vessel to wash the sample at any temperature, for example, at roomtemperature. A preferred immersion time is 3 minutes or more and 30minutes or less. PBS may be replaced with fresh one during the immersionas needed.

3) Staining with Fluorescent Particle Bonded to Biological SubstanceRecognition Site

A dispersion of the fluorescent particle bonded to a biologicalsubstance recognition site in PBS is placed on a tissue sample to reactwith a target biological substance. The type of the biological substancerecognition site bindable to the fluorescent particle can be varied tostain a variety of biological substances. In use of several types offluorescent particle bonded to different biological substancerecognition sites, these types of fluorescent particle bonded todifferent biological substance recognition sites in PBS may be premixed,or may be sequentially placed on the tissue sample.

The staining may be performed at any temperature, for example, at roomtemperature. A preferred reaction time is 30 minutes or more and 24hours or less.

Prior to the staining with the fluorescent particle, a known blockingagent, such as BSA-containing PBS, is preferably added dropwise to thesample.

The stained tissue sample is then immersed in PBS in a vessel to removeunreacted fluorescent particle. The unreacted fluorescent particle maybe removed at any temperature, for example, at room temperature. Apreferred immersion time is 3 minutes or more and 30 minutes or less.PBS may be replaced with fresh one during the immersion when necessary.The tissue sample is covered with a cover glass to seal the tissuesample. A commercially available sealant may be used when necessary.

In the case where staining with an HE staining reagent is performed, HEstaining is followed by the sealing of the tissue sample with the coverglass.

[Acquisition of Fluorescent Image]

A wide-field microscopic image (fluorescent image) of the stained tissuesample is taken with a microscopic imaging device 1A. In the microscopicimaging device 1A, an excitation light source and an optical filter fordetecting fluorescent light are selected according to the absorptionmaximum wavelength of the fluorescent substance used in the stainingreagent and the wavelength of the fluorescent light from the fluorescentsubstance.

<Operation of Pathological Diagnosis Support System 100>

The operation of the pathological diagnosis support system 100 forbiological substance quantitation (including staining, imageacquisition, and analysis described above) will now be described inreference to the flowchart in FIG. 5. Throughout the specification, theoperation will be described in an exemplary case in which a targetsample is a tissue sample stained with a staining reagent containing afluorescent particle bonded to a biological substance recognition sitethat can recognize a specific protein (HER2 protein in breast cancertissues in the present specification; hereinafter, referred to as aspecific protein) and in which a standard sample is plural kinds ofcells cultured on a substrate, such as a commercially availablemicroscopic slide, but should not be limited to this case. Any sample isincluded as long as it can be bonded to the fluorescent particle bondedto biological substance recognition site, such as biotin or antigen inwhich concentration of the biological substance is known.

First, an operator quantitates concentration of the specific protein inthe cultured cells, which is the standard sample of the presentembodiment (step S1). The concentration of the specific protein can bequantitated by any known method, for example, ELISA, flow cytometry,Western blotting, and the like. The concentration of the specificprotein per cell can be thereby calculated. According to ELISA andWestern blotting, the concentration of the specific protein can bequantitated from cells dissolved in a predetermined solution. Accordingto flow cytometry, biological molecules per cell can be detected andquantitated from cells scattered in a predetermined solution by lightscattering or fluorescence measurement with laser beam.

As a standard sample, the operator selects cultured cells in the samelot and having the same quality as the cultured cells in whichconcentration of the specific protein is quantitated. Any number ofkinds of standard samples may be selected. In order to obtain a highlyaccurate quantitation result by making calibration curves based on themeasurement result from the standard samples, plural kinds of standardsamples are preferably used. The plural kinds of standard samplespreferably have widely different concentration of specific proteinquantitated in step S1 are from each other.

Subsequently, the operator prepares formalin-fixed and paraffin-embeddedslices of a target sample(s) and standard samples (step S2) and thenstains the target sample and standard samples respectively under thesame staining conditions with two different kinds of staining reagent(i.e. HE staining reagent and a staining reagent including thefluorescent particle as a fluorescent labelling material, which isbonded with a biological substance recognition site recognizing thespecific protein) (step S3).

Staining under the same condition means that, for example, one operatorperforms staining process using staining reagents in the same lot, andthat the time, temperature, and humidity in each staining step aresubstantially constant. It is preferred that one operator performsstaining of the target sample and the standard samples sequentially andin parallel using the staining reagents in the same lot, so that thestaining conditions can be easily constant.

After that, a fluorescent image and a bright field image of each sample(the target sample and the standard samples) are respectively obtainedwith the microscopic image acquiring device 1A according to theprocedures (a1) to (a5). The images are input into the image processingdevice 2A and analyzed (step S4). The images of the target sample andthe standard samples are obtained and analyzed under the sameconditions.

-   (a1) The operator places each of the target sample and the standard    samples stained with the HE staining reagent and the staining    reagent containing the fluorescent particle on a microscopic slide,    and sets the slide on the slide fixation stage of the microscopic    image acquiring device 1A;-   (a2) The operator sets a bright field unit, and adjusts the    magnification for photographing and the focus so that the target    region of the tissue is in the field;-   (a3) The operator photographs the sample with the photographing unit    to generate image data of the bright field image, and transmits the    image data to the image processing device 2A;-   (a4) The operator replaces the bright field unit with a fluorescence    unit; and-   (a5) The operator photographs the sample with the photographing unit    without changing the field and the magnification to generate image    data of the fluorescent image, and transmits the image data to the    image processing device 2A.

Obtaining and analyzing images of the target sample and the standardsamples under the same conditions means that, specifically, thefluorescent image and the bright field image are respectively obtainedwith the same microscopic image acquiring device 1A under the sameconditions (for example, exposure time, magnification, white balance,and the like), and the cell image and the fluorescent bright spot imageare respectively extracted using the same threshold value and under thesame noise processing condition in each step in FIG. 6.

In the image processing device 2A, image analysis is performed on thebasis of the bright field image and the fluorescent image input from themicroscopic image acquiring device 1A. An evaluation value is measuredto show the quantitative evaluation of fluorescence from the fluorescentbright spots in a cell (step S4). In the present embodiment, afluorescent particle is used as fluorescent labelling material and thenumber of the fluorescent particle is determined as the evaluationvalue, but the present invention should not be limited to this. Forexample, integrated value of the fluorescent luminance may be determinedas the evaluation value.

FIG. 6 illustrates a detailed flowchart of the image analysis in stepS4. The image analysis illustrated in FIG. 6 is executed in cooperationwith the controller 21 and the program stored in the storage 25.

When the bright field image transmitted from the microscopic imageacquiring device 1A is input into the communication interface 24 (StepS401), a cell region is extracted from the bright field image (Step S402to step S405).

In extracting a cell region, the bright field image is converted into amonochromatic image (Step S402). FIG. 7A illustrates an exemplary brightfield image.

The monochromatic image is binarized using a predetermined thresholdvalue (Step S403).

In the next step, noise reduction process is performed (Step S404). Thenoise reduction can be performed, for example, by subjecting thebinarized image to a closing process. The closing process includesdilation process followed by erosion process executed as many times asthe dilation process. In the dilation process, a target pixel isreplaced with a white pixel if at least one white pixel is presentwithin the range of n×n pixels from the target pixel (where n is aninteger of 2 or more). In the erosion, the target pixel is replaced witha black pixel if at least one black pixel is present within the range ofn×n pixels from the target pixel. The closing process can remove smallregions such as noise. FIG. 7B illustrates an exemplary image after thenoise reduction process. An image of extracted cell regions (cell image)is obtained after the noise reduction process as in FIG. 7B.

In the next step, the image after the noise reduction process issubjected to labelling process to assign label to each of the extractedcells (Step S405). In the labelling process, the same label (number) isassigned to contiguous pixels in an image for identification of anobject. By the labelling process, the cells in the image after noisereduction can be identified and labelled.

Meanwhile, when the fluorescent image transmitted from the microscopicimage acquiring device 1A is input into the communication interface 24(Step S406: fluorescent image input step or standard fluorescent imageinput step), bright spot regions representing the presence of thefluorescent particle are extracted from the fluorescent image, and thefluorescent particle number in the bright spot regions is calculated(Steps S407 to S410: fluorescence quantitation step or standardfluorescence quantitation step).

At first, color components are extracted from the fluorescent image asin FIG. 8A, according to the emission wavelengths of the fluorescentbright spots (Step S407).

In Step S407, when the emission wavelength of the fluorescent particleis 615 nm, for example, only the fluorescent bright spots having thewavelength of 615 nm are extracted as an image. In the next step, theextracted image is subjected to a binarizing process using apredetermined threshold to generate a bright spot image of extractedbright spot regions (Step S408). FIG. 8B illustrates an exemplary brightspot image.

Noise removal process for removing autofluorescence of cells or otherunnecessary signal components may be executed prior to the binarizingprocess. A low-pass filter, such as a Gaussian filter, or a high-passfilter, based on a second derivative, is preferably used.

Subsequently, labelling process is executed to label each of theextracted bright spot regions (Step S409).

Subsequently, the fluorescent particle number in each extracted brightpoint region is calculated (step S410). The fluorescent particle numbermay be calculated by any method, for example, on the basis of theintegrated value of the luminance in each bright spot region.

After the process in step S410, the cell image (FIG. 7B) and the imageof extracted bright spot regions (FIG. 8B) are added (step S411), thedistribution of the bright spot regions in cells are shown at thedisplay 23 of the image processing device 2A, and the fluorescentparticle number per cell region is calculated (step S412).

After the process in step S412, the process returns to the steps in FIG.5 to prepare a distribution diagram and a calibration curve based on thedistribution diagram (step S5: correlation calculation step). Thedistribution diagram shows a correlation between the concentration ofthe specific protein in each standard sample measured in step S1 and thefluorescent particle number per cell in the standard sample measured instep S4, respectively plotted on a horizontal axis and on a verticalaxis.

Subsequently, the fluorescent particle number per cell in the targetsample calculated in step S4 is converted to the concentration ofspecific protein on the basis of the calibration curve prepared in stepS5 (step S6: conversion step).

According to the above-described present embodiment, the target samplewhich is an observation target and the standard samples in whichconcentration of the specific biological substance is quantitated inadvance are prepared through the processes in Steps S1 to S2. Stainingand image analysis under the same conditions are performed for thetarget sample and the standard samples through the processes in steps S3to S4. The fluorescent particle number in the target sample is convertedto the concentration of the specific protein through the processes insteps S5 to S6. Because the fluorescent particle number in the targetsample is converted to the concentration of the specific protein on thebasis of the measurement result for the standard sample measured underthe same conditions, the quantitation result of the specific protein canbe compared and evaluated even when at least one of the operator,constitution of the pathological diagnosis support system 100, and themeasurement conditions is different.

The above-descried embodiment is a suitable example of the presentinvention, and the present invention should not be limited to this.

For example, in step S6, the specific protein may be quantitated withoutpreparation of calibration curve. For example, the amount of thespecific protein can be calculated on the assumption that the ratio ofthe fluorescent particle number to the amount of the specific proteinper cell of the standard sample is equivalent to that of the targetsample. Thus, the quantitation of the specific protein can be easilyperformed on the basis of only one of standard sample. However,considering the accuracy in quantitating the specific protein, it ispreferred that the calibration curve is prepared on the basis of aplurality of standard samples.

It is generally known that the calibration curve for immunostainingshows a sigmoid shape, when it is prepared on the basis of the antigenconcentration plotted on a horizontal logarithmic axis and theevaluation value of staining on a vertical axis. Accordingly, thecalibration curve of the present invention prepared in step S6 in FIG. 5is preferably a sigmoid curve approximated to the distribution of theantigen concentration and the evaluation value of fluorescence. Morepreferably, only the most inclined portion of the prepared sigmoid curveis linearly approximated and used as a calibration curve. The range forthe linear approximation may be determined by any method. For example, acalibration curve may be determined as an approximate straight line onlywithin the range of the specific protein concentration at which thecorrelation coefficient of the approximate straight line is more than apredetermined value.

A sample of cultured cell is preferably used as a standard sample in thepresent embodiment because many homogenous samples can be easilyobtained so that there is strong correlation between the concentrationof the specific protein and the fluorescent particle number using manysamples. Accurate quantitation result can be thereby obtained.

In the present embodiment, the fluorescent substance used as a stainingreagent is described as a fluorescent particle in which a plurality ofmolecules of the fluorescent substance are accumulated, but may be onemolecule of fluorescent substance bonded to a biological substancerecognition site.

However, the high fluorescence from a fluorescent particle is hardlyaffected by noise due to photographic environment, such as environmentlight in the room or the efficiency of the image acquiring device.Furthermore, by using the fluorescent particle, not only the fluorescentluminance but the fluorescent particle number can be measured andquantitated from the fluorescent image. Furthermore, the luminance ofone particle is hardly affected by the time required for imageacquisition (for example, exposure time of excitation light) or thestorage condition of the stained samples, because the fluorescentparticle hardly photobleaches. Accordingly, the fluorescent particleyields less error in the evaluation result of fluorescence when used asa staining reagent, compared to the fluorescent substance which does notcompose a fluorescent particle. The highly accurate quantitation resultof the biological substance is thereby obtained. Considering the above,the fluorescent particle is preferably used as the staining reagent inthe present invention.

In step S5 of the present embodiment, the fluorescent particle number ismeasured per cell, however, it may be measured per cell nucleus or perimage area of the observation target, for example.

In the present embodiment, only one kind of specific protein isquantitated, however, plural kinds of specific protein may be stainedusing two or more fluorescent substances having different emissionwavelength from each other.

In such case, each of the color components is extracted using filters inStep S507, the processes in Steps S508 to S509 are executed for each ofthe extracted color components (wavelength components), and a cell imageand fluorescent particle images for each of the color components aresuperimposed in Step S11.

The fluorescent particle may be bonded to the biological substancerecognition site which bonds to a specific biological substance directlyas in the above embodiment or indirectly through other materials as inthe indirect method publically known in the field of immunostaining. Forexample, after the tissue sample is reacted with a primary antibodydirected against the specific antibody, the tissue sample is furtherreacted with a secondary antibody directed against the primary antibodyand bonded to a fluorescent particle so that the specific protein isstained. Otherwise, after the tissue sample is reacted with a primaryantibody directed against the specific antibody and further with abiotinylated secondary antibody directed against the primary antibody,the tissue sample is reacted with a fluorescent particle modified withstreptavidin. In this case, the specific protein is stained using thespecific bond of the streptavidin and the biotin to form a complex.

The description above discloses an example in which an HDD or asemiconductor nonvolatile memory is used as a computer-readable mediumfor the program according to the present invention, but the presentinvention should not be limited to this. Another computer-readablemedium may also be used, for example, a portable recording medium, suchas CD-ROM. Carrier waves can also be used as a medium that provides dataof the program according to the present invention through acommunication line.

The detailed configurations and the operations of the devices formingthe pathological diagnosis support system 100 can also be appropriatelymodified within the scope of the present invention.

The following VERIFICATION EXPERIMENTS 1 and 2 show the quantitationresults by conventional method (comparative examples) and thequantitation results obtained by correcting the comparative exampleswith the method of the present invention (present invention). Thestaining conditions (concentration of the staining reagent) for stainingKi67 protein in the tissue samples and the image acquiring condition(exposure time of excitation light (fluorescence)) were intentionallychanged in the experiments.

<Verification Experiment 1>

-   (A) Quantitation Using a Staining Reagent Including 0.02 Nm of    Fluorescent Particle-   (A-0) Preparation of Staining Reagent    [Preparation of Fluorescent Substance-Encapsulating Melamine    Nanoparticle]

Sulfo Rhodamine 101 (a red fluorescent dye made by Sigma-AldrichCorporation) (14.4 mg) as a fluorescent substance was dissolved in water(22 mL). A 5% aqueous solution (2 mL) of an emulsifier for emulsionpolymerization EMALGEN (registered trademark) 430 (polyoxyethylene oleylether, made by Kao Corporation) was added to the solution. The solutionwas heated to 70° C. with stirring on a hot stirrer, and 0.65 g of amelamine resin raw material NIKALAC MX-035 (made by NIPPON CARBIDEINDUSTRIES CO., INC.) was added to the solution.

A 10% aqueous solution (1000 μL) of dodecylbenzenesulfonic acid (made byKANTO CHEMICAL CO., INC.) as a surfactant was added to the solution, andwas stirred at 70° C. for 50 minutes. The solution was heated to 90° C.,and was stirred for 20 minutes at the temperature. The obtaineddispersion of resin particle with dye was washed with pure water toremove impurities, such as excess resin raw material and excessfluorescent dye.

Specifically, the dispersion was centrifuged with a centrifuge (MicroCooling Centrifuge 3740 made by Kubota Corporation) at 20000 G for 15minutes and the supernatant was removed. Ultrapure water was added, andthe solution was redispersed by ultrasonic waves. The centrifugation,removal of the supernatant, and redispersion in ultrapure water wasrepeated five times. The obtained melamine particle was positivelycharged, due to a lot of amino groups in the skeleton of the melamineresin. The evaluation of charge of the resin particle was performed bycomponent analysis of resin by NMR, IR, and the like, and by measurementof zeta potential.

The obtained nanoparticle 1 was observed with a scanning electronmicroscope (SEM; S-800 made by Hitachi, Ltd.). The average particle sizewas 150 nm and the coefficient of variation was 12%.

[Bonding of Antibody to Fluorescent Particle]

An anti-Ki-67 antibody was bonded to the fluorescent particle by thefollowing Steps (1) to (12).

-   Step (1): Disperse 1 mg of the fluorescent particle 1 in 5 mL of    pure water. Next, add 100 μL of an aminopropyltriethoxysilane    aqueous dispersion (LS-3150; Manufactured by Shinetsu Kagaku Co.,    Ltd.) thereto and perform stirring for 12 hours at room temperature.-   Step (2): Perform centrifugation on the reacted mixture at 10,000 G    for 60 minutes and remove the supernatant.-   Step (3): Add ethanol and disperse the precipitates and perform    centrifugation again. By the same procedure, perform washing with    ethanol once and washing with pure water once.

The obtained fluorescent particle modified with the amino group wassubjected to FT-IR measurement. Adsorption derived from the amino groupwas observed, and it was confirmed that the particle had been modifiedwith the amino group.

-   Step (4): Adjust the fluorescent particle modified with the amino    group obtained in Step (3) to 3 nM using PBS containing 2 mM of EDTA    (ethylenediaminetetraacetic acid).-   Step (5): Mix the adjusted solution in Step (4) with SM(PEG) 12    (Thermo Scientific,    succinimidyl-[(N-maleomidopropionamid)-dodecaethyleneglycol]ester)    so that the final concentration is 10 mM, and perform reaction for 1    hour.-   Step (6): Perform centrifugation on the reacted mixture at 10,000 G    for 60 minutes and remove the supernatant.-   Step (7): Add PBS containing 2 mM of EDTA to disperse the    precipitates and perform centrifugation again. With the same    procedure, perform washing three times. Finally, perform    re-dispersion by using 500 μL of PBS.-   Step (8): Dissolve 100 μg of an anti Ki67 antibody in 100 μL of PBS,    add 1M dithiothreitol (DTT) thereto, and make it react for 30    minutes.-   Step (9): Remove excessive DTT from the reacted mixture with a gel    filter column to obtain a reduced anti Ki67 antibody solution.-   Step (10): Mix the particle dispersion obtained at Step (7) with the    fluorescent particle 1 as the starting material with the reduced    anti Ki67 antibody solution obtained at Step (9) in PBS, and make it    react for 1 hour.-   Step (11): Add 4 μL of 10 mM mercaptoethanol so as to end the    reaction.-   Step (12): Perform centrifugation on the reacted mixture at 10,000 G    for 60 minutes, remove the supernatant, and then add PBS containing    2 mM of EDTA so as to disperse the precipitates and perform    centrifugation again. With the same procedure, perform washing three    times. Finally, perform re-dispersion by using 500 μL of PBS,    thereby obtaining the fluorescent particle with anti Ki67 antibody    bonded.    (A-1) Preparation of Target Sample and Standard Sample

Three spots ((a), (b), and (b)) were selected in a breast cancer array.The breast cancer array was cut into slices and determined to be targetsamples after fixing, dehydrating, and embedding the slices.

The expression amount of Ki67 protein was measured with ELISA kit (HumanHER2 (Total) kit, No. KH00701 made by Invitrogen Corporation) from thecommercially available five kinds of cultured cell lines (CRL1500,NDA-MB175, COLO201, Hela, and NDA-MB231). The cultured cell linesrespectively in the same lot as the above five kinds of cultured celllines were determined as standard samples.

(A-2) Staining of Tissue with Fluorescent Particle

Immunostaining of target samples and standard samples was performed bythe following

-   Steps (1) to (11). The same operator performed the immunostaining of    the target samples and standard samples in parallel, so that the    immunostaining was performed almost at the same time and under the    same conditions.-   Step (1): Immerse the target sample or the standard sample in a    container containing xylene for 30 minutes. Change the xylene three    times during the immersion.-   Step (2): Immerse the target sample or the standard sample in a    container containing ethanol for 30 minutes. Change the ethanol    three times during the immersion.-   Step (3): Immerse the target sample or the standard sample in a    container containing water for 30 minutes. Change the water three    times during the immersion.-   Step (4): Immerse the target sample or the standard sample in 10 mM    citric acid buffer solution (pH 6.0) for 30 minutes.-   Step (5): Perform autoclaving for 10 minutes at 121 degrees.-   Step (6): Immerse the target sample or the standard sample in a    container containing PBS for 30 minutes.-   Step (7): Put 1% BSA-containing PBS on the target sample or the    standard sample and leave it as it is for 1 hour.-   Step (8): Put the staining reagent diluted with 1% BSA-containing    PBS to 0.02 nM on the target sample or the standard sample and leave    it as it is for 3 hours.-   Step (9): Immerse the target sample or the standard sample in a    container containing PBS for 30 minutes.-   Step (10): Perform hematoxylin staining after fixation with 4%    neutral Paraformaldehyde solution for 10 minutes.-   Step (11): Drip Aquatex, produced by Merck Chemicals, thereon and    then place a cover    (A-3) Acquiring Microscopic Image

With respect to each of the target samples and the standard samples,microscopic images (a bright field image and a fluorescence image) wereacquired.

As a microscope, an upright microscope Axio Imager M2 (produced by CarlZeiss AG) was used. The objective lens was set to 20 times. In obtaininga fluorescence image, each tissue section was irradiated with excitationlight having a wavelength of 580 nm for exposure time of 200 mn, animage of fluorescence having a wavelength of 610 nm emitted from thetissue section was formed, and a microscopic image (image data) wasacquired with a camera (monochrome) set in the microscope. The camerahas 6.4 μm×6.4 μm as the pixel size, 1,040 pixels as the number ofpixels in height and 1,388 pixels as the number of pixels in width (acapturing region of 8.9 mm×6.7 mm).

The fluorescent images of the target samples and the standard sampleswere obtained almost at the same time and under the same conditions bythe same operator with the same apparatus.

(A-4) Calculation of Fluorescent Particle Number Per Cell

The microscopic images of the target samples and the standard samplesobtained in (A-3) were subjected to image analysis illustrated in FIG. 6and the fluorescent particle number per cell was calculated as anevaluation value of fluorescence. In calculating the fluorescentparticle number, a binary image was prepared based on a predeterminedhigher threshold and a lower threshold. The fluorescent particle numberwas counted with bright spot measuring software “G-count”made byG-Angstrom K.K. After that, the bright field image obtained in (A-3) andthe fluorescent image was superimposed. The number of bright spots incell regions were calculated and thus the fluorescent particle numbersin a cell were calculated for the target samples and the standardsamples, respectively. The fluorescent particle number in a cell of thetarget sample (fluorescent particle number/cell) is determined asCOMPARATIVE EXAMPLE 1.

(A-5) Quantitation of Expression Amount of Ki67 Protein in One Cell

As shown in FIG. 9, the distribution diagram was prepared. In thediagram, the horizontal axis represents the expression amount of Ki67protein previously measured from the cultured cell line in the same lotas the standard sample. The vertical axis represents the fluorescentparticle number in a cell of the standard sample calculated in (A-4).Dotted lines in FIG. 9 are calibration curves obtained by linearapproximation of the distribution. The fluorescent particle number in acell of the target samples was converted to the concentration (ng/ml) ofKi67 protein in a cell of the target samples on the basis of thecalibration curves and was determined as PRESENT INVENTION 1.

(B) Quantitation Using a Staining Reagent Including 0.06 Nm ofFluorescent Particle

The fluorescent particle number per cell was quantitated in each of thetarget samples and the standard samples as in the same processes as theabove-described (A), except that staining reagent after dilutioncontains 0.06 nM of the fluorescent particle in step (8) of theabove-described (A-2). The fluorescent particle number in a cell(fluorescent particle number/cell) of the target sample was determinedas COMPARATIVE EXAMPLE 2. The fluorescent particle number in a cell ofthe target sample converted to the concentration of Ki67 protein (ng/ml)in a cell of the target samples was determined as PRESENT INVENTION 2.

Among the slices of the three spots ((a) to (c)) of the breast cancerarray described in above (A-1), those adjacent to the three slicesstained and used for quantitating Ki67 protein concentration in (A) wasused as the target samples.

(C) Comparison of Quantitation Result with Different Concentration ofStaining Reagent

FIG. 9 shows calibration curves prepared in the quantitation stepdescribed in above (A) and (B). Each calibration curve was made bylinear approximation of the fluorescent particle number in thefluorescent image plotted against the Ki67 protein concentrationquantitated by ELISA for the five standard samples. While the gradientof the calibration curve was about 0.69 when the fluorescent particleconcentration was 0.02 nM, it was about 1.85 when the fluorescentparticle concentration was 0.06 nM. That is, the gradients were aboutthree times different.

TABLE-1 shows the quantitation results of PRESENT INVENTIONS 1 and 2 andCOMPARATIVE EXAMPLES 1 to 2.

TABLE 1 CONCENTRATION OF FLUORESCENT SPOT SPOT SPOT PARTICLE [nM] (a)(b) (c) PRESENT 0.02 5.1 10 40.7 INVENTION 1 PRESENT 0.06 6.6 11 43.5INVENTION 2 COMPARATIVE 0.02 4.8 8.2 29.3 EXAMPLE 1 COMPARATIVE 0.06 1826.2 86.3 EXAMPLE 2

When the concentration of the fluorescent particle in the stainingreagent increased by about three times, the quantitation resultsincreased by about 2.4 to 3.1 times in all the spots (a) to (c)according to COMPARATIVE EXAMPLES 1 and 2, in which the quantitationresults were the fluorescent particle number in a cell quantitated bythe conventional method and without correction using calibration curves.

Meanwhile, even when the concentration of the fluorescent particle inthe staining reagent increased by about three times, the quantitationresults were about 0.77 to 0.94 times according to PRESENT INVENTIONS 1and 2, in which the fluorescent particle number was converted to theconcentration of the protein using calibration curves of FIG. 9 by themethod of the present invention. The difference in quantitation resultscaused by the different concentration of the staining reagent wasreduced by correction on the basis of calibration curves by the methodof the present invention.

<Verification Experiment 2>

(D) Quantitation with Exposure Time of 100 Ms in Fluorescent ImageAcquisition

From the samples prepared and stained in above (A-1) and (A-2), thefluorescent particle number per cell was quantitated in each of thetarget samples and the standard samples in the same processes as theabove-described (A-3) to (A-5), except that exposure time of excitationlight was 100 ms in the above (A-3). The fluorescent particle number inone cell (fluorescent particle number/cell) of the target sample wasdetermined as COMPARATIVE EXAMPLE 3. The fluorescent particle number ina cell of the target samples converted to the concentration of Ki67protein (ng/ml) in a cell of the target samples was determined asPRESENT INVENTION 3.

TABLE-2 shows the quantitation results of PRESENT INVENTIONS 1 and 3 andCOMPARATIVE EXAMPLES 1 and 3.

TABLE 2 EXPOSURE SPOT SPOT SPOT TIME [ms] (a) (b) (c) PRESENT INVENTION1 100 5.1 10 40.7 PRESENT INVENTION 3 200 6 9.3 38.4 COMPARATIVE 100 4.88.2 29.3 EXAMPLE 1 COMPARATIVE 200 2.4 3.6 14.2 EXAMPLE 3

When the exposure time in fluorescent image acquisition was about 0.5times, the quantitation results was about 0.5 to 0.57 times in any ofthe spots (a) to (c) according to COMPARATIVE EXAMPLES 1 and 3, in whichthe quantitation result was the fluorescent particle number in a cellquantitated by the conventional method and without correction usingcalibration curves.

Meanwhile, even when the exposure time in fluorescent image acquisitionwas about 0.5 times, the quantitation results were about 0.85 to 1.08times according to PRESENT INVENTIONS 1 and 2, in which the fluorescentparticle number was converted to the concentration of the protein by themethod of the present invention as in the VERIFICATION EXPERIMENT 1. Thedifference in quantitation results caused by the different exposure timein fluorescent image acquisition was reduced by correction on the basisof calibration curves by the method of the present invention. Althoughembodiments of the present invention have been described and illustratedin detail, it is clearly understood that the same is by way ofillustration and example only and not limitation, and the scope of thepresent invention should be interpreted by terms of the appended claims.

INDUSTRIAL APPLICABILITY

A specific biological substance in a tissue sample can be accuratelyquantitated by the present invention. The present invention can beparticularly preferably applied in generating highly accurateinformation for pathological diagnosis.

DESCRIPTION OF REFERENCE NUMERALS

-   1A microscopic image acquiring device-   2A image processing device-   3A cable-   21 controller (fluorescence quantitating unit, a standard    fluorescent quantitation unit, a correlation calculation unit, a    conversion unit)-   22 operation unit-   23 display-   24 communication interface (fluorescent image inputting unit,    standard fluorescent image inputting unit)-   25 storage-   26 bus-   100 pathological diagnosis supporting system

The invention claimed is:
 1. A biological substance quantitation methodwhich quantitates an expression amount of a specific biologicalsubstance in a sample stained with a staining reagent which stains thebiological substance with a fluorescent substance, the methodcomprising: obtaining a fluorescent image of the sample; inputting thefluorescent image which represents an expression of the biologicalsubstance in the sample by a fluorescent bright spot; performingfluorescence quantitation which includes calculation of an evaluationvalue by quantitative evaluation of the fluorescent bright spot in thefluorescent image; inputting a standard fluorescent image under a samecondition as the inputting of the fluorescent image, wherein thestandard fluorescent image represents an expression of the biologicalsubstance in a standard sample by a fluorescent bright spot based onstaining under a same condition as the sample, and an expression amountof the biological substance in the standard sample is measured inadvance; performing standard fluorescence quantitation which includescalculation of an evaluation value by quantitative evaluation of thefluorescent bright spot in the standard fluorescent image under a samecondition as the quantitation of fluorescence; calculating a correlationbetween the expression amount of the biological substance in thestandard sample and the evaluation value of the fluorescent bright spotin the standard fluorescent image; and converting the evaluation valueof the fluorescent bright spot in the fluorescent image to an expressionamount of the biological substance in the sample based on thecalculating of the correlation.
 2. The biological substance quantitationmethod according to claim 1, wherein a calibration curve is prepared incalculating the correlation, wherein the calibration curve representsthe evaluation value of the fluorescent bright spot of the standardfluorescence image corresponding to the expression amount of thebiological substance in the standard sample, and in the converting, theevaluation value of the fluorescent bright spot of the fluorescent imageis converted to the expression amount of the biological substance in thesample based on the calibration curve.
 3. The biological substancequantitation method according to claim 1, wherein the standard sample isa cell cultured on a substrate.
 4. The biological substance quantitationmethod according to claim 1, wherein the biological substance is aprotein.
 5. The biological substance quantitation method according toclaim 1, wherein the staining reagent comprises a fluorescent particlein which a plurality of molecules of the fluorescent substance areaccumulated.
 6. A pathological diagnosis support system whichquantitates an expression amount of a specific biological substance in asample stained with a staining reagent which stains the biologicalsubstance with a fluorescent substance, the system comprising: a storagewhich stores a program for quantitating the amount of the specificbiological substance; and a processor, the program causing the processorto function as: a fluorescent image inputting unit which inputs afluorescent image which represents an expression of the biologicalsubstance in the sample by a fluorescent bright spot; a fluorescencequantitation unit which calculates an evaluation value by quantitativeevaluation of the fluorescent bright spot in the fluorescent image; astandard fluorescent image inputting unit which inputs a standardfluorescent image under a same condition as the fluorescent imageinputting unit, wherein the standard fluorescent image represents anexpression of the biological substance in a standard sample by afluorescent bright spot based on staining under a same condition as thesample, and an expression amount of the biological substance in thestandard sample is measured in advance; a standard fluorescentquantitation unit which calculates an evaluation value by quantitativeevaluation of the fluorescent bright spot in the standard fluorescentimage under a same condition as the fluorescence quantitation unit; acorrelation calculator which calculates a correlation between theexpression amount of the biological substance in the standard sample andthe evaluation value of the fluorescent bright spot in the standardfluorescent image; and a converter which converts the evaluation valueof the fluorescent bright spot in the fluorescent image to an expressionamount of the biological substance in the sample based on thecorrelation.
 7. A non-transitory recording medium storing a computerreadable program causing a computer which quantitates an expressionamount of a specific biological substance in a sample stained with astaining reagent which stains the biological substance with fluorescentsubstances, to function as obtaining a fluorescent image of the sample:a fluorescent image inputting unit which inputs a fluorescent imagewhich represents an expression of the biological substance in the sampleby a fluorescent bright spot; a fluorescence quantitation unit Whichcalculates an evaluation value by quantitative evaluation of thefluorescent bright spot in the fluorescent image; a standard fluorescentimage inputting unit which inputs a standard fluorescent image under asame condition as in the fluorescent image inputting unit, wherein thestandard fluorescent image represents an expression of the biologicalsubstance in a standard sample by a fluorescent bright spot based onstaining under a same condition as the sample, and an expression amountof the biological substance in the standard sample is measured inadvance; a standard fluorescent quantitation unit which calculates anevaluation value by quantitative evaluation of the fluorescent brightspot in the standard fluorescent image under a same condition as thefluorescence quantitation unit; a correlation calculator whichcalculates a correlation between the expression amount of the biologicalsubstance in the standard sample and the evaluation value of thefluorescent bright spot in the standard fluorescent image; and aconverter which converts the evaluation value of the fluorescent brightspot in the fluorescent image to an expression amount of the biologicalsubstance in the sample based on the correlation.
 8. The biologicalsubstance quantitation method according to claim 1, wherein theexpression amount of the biological substance in the standard sample ismeasured in advance by one of ELISA, flow cytometry, and Westernblotting.